The present invention relates to methods for processing animal tissue, particularly methods for dispersing decellularized human skin to produce injectable tissue matrix compositions.
Collagen is one of the most abundant proteins in the animal kingdom and is the primary structural component of connective tissues, such as skin, ligaments, tendons, and bones. Collagen possesses many properties, including high tensile strength, low immunogenicity, semipermeability, and solubility, that make it particularly suitable for use in the preparation of various biomaterials and other medical products, including medical prostheses, implantable compositions, cellular and acellular grafts, and other tissue replacement materials. Recently, injectable collagen formulations have been widely used in a variety of applications, including as tissue bulking compositions and as ophthalmic implants.
Collagen compositions are often prepared from skin, ligaments, or tendons by dispersion, digestion and/or dissolution. Dispersion typically involves mechanically shearing the tissue to produce a crude homogenate tissue matrix. Tissue digestion and dissolution generally entails enzyme degradation of a portion of the non-helical telopeptide portions of the collagen molecules, followed by purification steps, to produce a solution of telopeptide-poor collagen. The solubilized collagen can be reconstituted into molecular aggregates and occasional fibrils by neutralizing the enzyme digested, purified collagen solution.
Although it is generally desirable to use autologous tissues, i.e., those derived from the recipient of the implant, in the preparation of injectable collagen-containing compositions, there is seldom an ample supply of transplantable autologous tissues. Thus, allograft tissues and xenograft tissues are often used. Unfortunately, allogeneic and xenogenic tissues may be rejected by the recipient organism and may be associated with a greater risk of disease transmission. However, approximately 450,000 allograft tissues are transplanted each year in the U.S., and during the past decade, improvements in donor screening and serological testing have greatly reduced the risk of infectious disease transmission. For example, there have been no confirmed transmissions of AIDS through tissue transplants distributed by the Musculoskeletal Transplant Foundation, Edison, N.J., (MTF) in more than 10 years following the development of sensitive and accurate test methods. The estimated odds of contracting the AIDS virus from a transplant are less than 1 in 1.67 million (annual), without the inclusion of viral inactivation and sterilization steps. However, allograft implants may still be rejected following implantation. Allograft skin, used to treat burns, eventually becomes rejected, primarily due to allorecognition of Class II MHC antigens associated with Langerhans cells present in the epidermal layer of skin.
Thus, there is a need for improved methods for preparing injectable collagen compositions, particularly methods that are able to make use of allogeneic and xenogenic tissue sources while avoiding the complications that often accompany implantation of such materials.
The present invention features a method for processing dermal tissue for implantation into a subject. The method includes the steps of: (a) removing the epidermal layer of the dermal tissue to produce de-epidermalized tissue; (b) incubating the de-epidermalized tissue in at least one processing solution to remove cells from the de-epidermalized tissue, thereby producing a decellularized tissue matrix; and (c) exposing the decellularized tissue matrix to an acylating agent, wherein the ratio of acylating agent to wet tissue weight is about 0.003:1 or less. In a particularly preferred embodiment, the decellularized tissue matrix is treated, e.g., by cryomilling, to increase its surface area prior to acylation. This processing using low levels of acylating agent combined with cryomilling consistently results in relatively high yields of dispersed tissue matrix having a high resistance to trypsin.
In one embodiment of the invention, the dermal tissue is de-epidermalized by exposing the dermal tissue to a hypertonic salt solution, which allows for separation of the dermis and epidermis. The de-epidermalized tissue may then be processed by incubation in a variety of processing solutions. For example, the de-epidermalized tissue is preferably incubated in a series of decellularization solutions, including a high pH (e.g., sodium hydroxide) solution, a low pH (e.g., hydrochloric acid or phosphoric acid) solution, and a solvent (e.g., reagent alcohol). Incubation in such solutions may be in any order. For example, the de-epidermalized tissue may be incubated in low pH solutions first and then in high pH solutions and solvent solutions (or vice versa). In addition to removing cells from the tissue, this process also leads to inactivation of viruses and other contaminants in the tissue. Optionally, the tissue may be exposed to any of a number of viral inactivating agents before, during, or after the decellularization process.
In another related aspect, the invention provides a method for dispersing decellularized animal tissue, which method involves contacting any type of decellularized animal connective tissue with a solution comprising an acylating agent, wherein the ratio of acylating agent to wet tissue weight is about 0.003:1 or less. As discussed above, the decellularized tissue may be treated, for example by cryomilling, to increase the surface area of the tissue prior to decellularization.
The tissues processed according to the methods of the invention may come from autogenic, allogeneic, or xenogenic sources. In various preferred embodiments, the tissue is mammalian, preferably human; the acylating agent is an anhydride such as glutaric anhydride or succinic anhydride; and the ratio of acylating agent to wet tissue weight is within the range of about 0.002:1 to about 0.001:1.
The methods of the invention can be used to produce injectable, dispersed collagen compositions that have a trypin resistance greater than about 40%, preferably greater than about 50%, more preferably greater than about 70%, and most preferably than 90%. Preferably, the dispersed collagen matrix compositions of the invention are injectable through needles as small as 30 gauge.
In another aspect, the invention features a method of using the compositions of the invention for altering the condition of in situ tissue of a mammalian subject. The method involves placing an effective amount of the composition, e.g., as an injectable flowable mass, or formed into a putty like spreadable mass or finely divided distributable particles, at the in situ tissue site to be altered.
Other advantages and features of the present invention will be apparent from the following detailed description thereof and from the claims.
By xe2x80x9cacylating agentxe2x80x9d is meant an agent that transfers an acyl group to another nucleophile. Examples of acylating agents include anhydrides, acid chlorides, sulfonyl chlorides, and sulfonic acids.
The terms xe2x80x9cautologousxe2x80x9d and xe2x80x9cautogenicxe2x80x9d refer to tissues or cells which originate with or are derived from the recipient, whereas the terms xe2x80x9callogeneicxe2x80x9d and xe2x80x9callograftxe2x80x9d refer to tissues or cells which originate with or are derived from a donor of the same species as the recipient. The terms xe2x80x9cxenogenicxe2x80x9d and xe2x80x9cxenograftxe2x80x9d refer to tissues or cells that originate with or are derived from a species other than that of the recipient.
By xe2x80x9ccryomillingxe2x80x9d is meant a reduction in size by homogenizing or pulverizing the tissue in the presence of liquid nitrogen or other such solutions that cause the tissue to remain in a frozen state during the homogenizing or pulverizing process.
By xe2x80x9cdecellularized tissuexe2x80x9d is meant tissue that is substantially free of cells or cellular debris (i.e. a substantially acellular tissue matrix) as determined by light microscopy or by biochemical methods capable of identifying cells or cellular debris. By xe2x80x9csubstantially acellularxe2x80x9d is meant having at least 95% fewer native cells and cell structures than the natural state of the tissue as determined by light microscopy or by biochemical methods capable of identifying cells or cellular debris.
By xe2x80x9cprocessing solutionxe2x80x9d is meant a solution that is capable of inactivating viral or bacterial contaminants contained within animal tissue and/or is capable of removing cells within animal tissue while preserving the extracellular tissue matrix.
By xe2x80x9cde-epidermalized tissuexe2x80x9d is meant a portion of skin from which the epidermis has been substantially removed.
By xe2x80x9cimplantxe2x80x9d is meant any biomaterial that is introduced into the body of a patient to replace or supplement the structure or function of the endogenous tissue.
By xe2x80x9cdermalxe2x80x9d is meant of or relating to or located in the skin. By xe2x80x9cdermisxe2x80x9d is meant layers of the skin underlying the epidermis and overlying the subcutaneous structures.
By xe2x80x9csubjectxe2x80x9d is meant a mammalian organism, preferably a human or other primate species.
By xe2x80x9ctissuexe2x80x9d is meant an aggregation of similarly specialized cells in an organism, preferably, mammalian, and, most preferably, human, where the cells are exposed to the organism""s extracellular fluid, and are united in performance of a function within an organism.
By xe2x80x9ctrypsin resistancexe2x80x9d is meant the ability of the dispersed tissue matrix to resist digestion bytrypsin. When trypsin resistance is stated in terms of a percentage, this refers to the amount of tissue which remains undigested when exposed to 2% trypsin at 37xc2x0 C. for 6-24 hours.
The present invention provides methods for processing animal tissues, particularly methods for preparing decellularized tissue in a form that is injectable through a cannula or through needles as small as 30 gauge. The process of the invention inactivates viral loads in tissues and effectively decellularizes tissue. Furthermore, the process produces a decellularized, injectable tissue matrix composition with high yields and with high resistance to digestion by non-collagenase proteases, such as trypsin.
The invention features methods for processing connective (collagenous) tissues, such as intact skin, from autogenic, allogenic, or xenogenic sources into a dispersed form. In the case of allogenic and xenogenic skin, methods are provided to remove epidermis, to inactivate viruses, to decellularize tissue, and to disperse the tissue via an acylation reaction into a composition retaining the characteristics and functionality of the major tissue matrix components, i.e., collagen types and collagen fibrils, elastic fiber network, and most proteoglycans, glycoproteins, and glycosaminoglycans. In addition, the dispersed matrix compositions exhibit high resistance to digestion by non-collagenase proteases and can be produced at high yields. These important properties result in part from a novel method step in which the comminuted dermis is further reduced in size by a cryomilling process prior to acylation. This cryomilling step increases the total surface area of tissue available for the acylation reaction, thereby allowing for a reduction in the amount of acylating agent needed for dispersion while maintaining high yields.
The methods of the invention may be used in processing any type of connective (collagenous) tissue from autogenic, allogeneic, or xenogenic sources. While autologous tissues (transplanted from one location to another in the individual""s own body) are generally regarded as the safest of all implant materials, they are not always available. Thus, allograft tissue (transplanted from one individual to another) is a preferred alternative choice for implant materials. Xenogenic sources may also be used.
The tissues used in the present invention may be derived, for example, from human, bovine, porcine, canine, ovine, caprine, equine, or other mammalian organisms. Tissue structures such as dermis, artery, vein, pericardium, heart valve, dura mater, ligament, intestine, and fascia may all be subjected to the processing techniques described herein to yield an acellular tissue matrix composition.
Human tissue for processing according to the methods of the present invention may be obtained from tissue banks or directly from hospitals. Animal tissues are obtainable from a number of meat processing companies and from suppliers of laboratory animals. Ideally, tissue procurement procedures are selected to minimize disruption of the extracellular tissue matrix or other mechanical or biochemical damaging events. The harvested tissue may be processed while still fresh or may be stored in a freezer for later processing.
While the present invention can be used in processing any type of connective tissue from any animal source, the preferred tissue for processing is human skin. To reduce or eliminate allorecognition of skin, it is important to remove the epidermis housing Langerhans cells. The epidermis is easily removed using a number of well-known techniques (see Skerrow and Skerrow, Chapter 23 xe2x80x9cA survey of Methods for the Isolation and Fractionation of Epidermal Tissue and Cellsxe2x80x9d In: Methods of Skin Research, Eds: D. Skerrow and C. J. Skerrow, John Wiley and Sons, Ltd., 1985, Pp. 609-650). For example, incubation of the skin in a hypertonic saline solution (2M NaCl) will allow for the clean separation of the epidermis and dermis without damaging the extracellular tissue matrix. The de-epidermalized skin can then be cut or macerated into appropriately sized sections or pieces for further processing.
In the present invention, the connective tissue (e.g., dermis) is preferably incubated in one or more processing solutions in order to inactivate viruses and other contaminants (such as bacterial or other microbial contaminants) and to remove cells (including epithelial cells, endothelial cells, smooth muscle cells, and fibroblasts) from the tissue while maintaining the structural integrity of the extracellular (e.g., collagenous) tissue components, thereby producing a decellularized tissue matrix. A variety of well-known chemical, biochemical, and physical methods may be used to accomplish decellularization. These techniques include, but are not limited to, incubation in certain salts, detergents, or enzymes, radiation exposure, vapor freezing, hypotonic lysis, and treatment with acidic and/or alkaline solutions (see, e.g., U.S. Pat. Nos. 5,192,312, 5,336,616, 5,993,844, 5,613,982). Whatever decellularization technique is employed, it preferably should not disrupt or substantially alter the biomechanical properties of the structural elements of the tissue. In a preferred embodiment, the tissue is treated with a high pH solution, followed by treatment in a low pH solution and then by treatment is a solvent solution, thereby inactivating viral contaminants and removing cells from the tissue. This approach has the advantage of producing a substantially acellular tissue matrix that is free of detergents, enzymatic modifications, or other unwanted effects that can be associated with other methods of decellularization.
Methods for preparing and utilizing tissue compositions that include implantable, cross-linkable, telopeptide-containing, naturally crosslinked human collagen derived from comminuted intact human tissue are well-known in the art. (See, e.g., U.S. Pat. Nos. 4,969,912 and 5,332,802, both of which are incorporated herein by reference.) Such compositions are prepared by reacting comminuted tissues with an amine acylating agent or a carboxylic reactive esterifying agent or combination thereof at weight ratios of amine reactive acylating agent to wet tissue of from about 0.005:1 to about 0.5:1. Such preparations can be utilized for altering the condition of autogenic or allogenic in situ tissue.
In the present invention, dispersion of decellularized tissue is accomplished via an acylation step. Without being bound to a particular theory, we posit that such dispersion occurs as the acylating agent reacts with nonspecific, deprotonated proteins binding matrix collagen fibrils together in fibers and fiber bundles. Some acylation of collagen fibrils also occurs. Excess acylation of collagen matrix structures causes destabilization of such reacted collagen fibrils resulting in sensitivity to digestion by noncollagenase proteases, such as trypsin. Trypsin generally is ineffective at digesting intact collagen structures. The acylation reaction is also rapid, probably complete within 1 minute. Since acylating reagents are water sensitive, they rapidly undergo hydrolysis into acid forms. Thus, there is competition between reacting with deprotonated amines and undergoing hydrolysis. The acylation reaction must be carefully controlled to allow enough reactivity to disperse the tissue matrix but not to allow too much reactivity such that the collagen fibril matrix is destabilized and subject to digestion by noncollagenase proteases.
We have found that the dispersion reaction can be optimized to allow effective tissue dispersion while maintaining the integrity and structure of the collagenous matrix. In the present invention, this is accomplished by increasing the surface area of the tissue prior to acylation and by decreasing the ratio of amine acylating agent to wet tissue to levels of about 0.004:1 or less, preferably less than 0.003:1, and most preferably from about 0.002:1 to about 0.001:1. As shown in the Examples below, dispersed dermis prepared using such improvements exhibit significantly higher yields, higher resistance to trypsin, and greater clinical longevity than dispersed dermis prepared using previously described methods (for example, U.S. Pat. No. 5,332,802). Typically, dispersed dermis prepared by the methods of the invention exhibit a trypsin resistance greater than about 40%, preferably greater than about 50%, most preferably greater than about 70% or 90%.
In order to increase the surface area of the tissue prior to acylation, the processed (decellularized) tissue is preferably subjected to a cryomilling process, which involves, for example, placing coarsely macerated treated dermis in a stainless steel container containing a level of liquid nitrogen approximately 1 inch over the tissue; holding the tissue in a frozen state for at least 10 minutes; macerating the frozen tissue for several bursts of 1-3 minutes each in a maceration chamber using a static rotor/stator macerator/size reduction apparatus while maintaining the macerated tissue in a frozen state by adding more liquid nitrogen. An example of such an apparatus is the TEKMAR(copyright) Analytical Mill for milling dry or frozen tissues. Alternatively, other techniques for increasing the surface area of the processed tissue may be employed, such as mincing the tissue, microfluidization (forcing coarse macerated tissue through sequentially smaller diameter cannula to decrease particle size), sonication, and other such methods to reduce particle size. Increasing the surface area of the tissue prior to acylation increases the effectiveness of the dispersion process, thus allowing lower amounts of acylating agent to be used while still achieving relatively high product yields. This reduction in the amount of acylating agent, in turn, leads to greater trypsin resistance in the dispersed tissue.
A preferred acylating reaction for the dispersion of processed (decellularized) tissue is acylation with glutaric anhydride or succinic anhydride. However, any acylating agent may be used for the dispersion reaction, for example, any anhydride, acid chloride, sulfonyl chloride, or sulfonic acid may be used. All of these acylating agents work by reacting with, and derivatizing, the deprotonated free amines of a protein. In many cases, the free amine group is derivatized with a chemical moiety that provides an anionic group, thereby reducing the pKa of the protein. For certain proteins, such as collagen, this also renders the protein more soluble at physiological pH.
In general, the acylating agent may be an aliphatic or aromatic, mono-, di-, or higher functional carboxylic acid anhydride, ester or halide; or sulfonic acid or halide, such as a lower alkanoic, lower alkane-dioic, or higher functional lower alkane carboxylic, or aryl mono-, di-, or higher functional carboxylic (e.g., benzoic or naphthoic), acid anhydride, ester or halide, or lower alkyl, or aryl (e.g., phenyl or naphthyl), mono-, di-, or higher functional sulfonic acid or halide, to provide the corresponding acyl (carbonyl or sulfonyl) moiety on the amine group, for example, lower alkanoyl, aroyl (e.g., phenoyl or naphthoyl), alkyl sulfonyl, aryl (e.g., phenyl or naphthyl), sulfonyl, or substituted amino (amido or sulfonamido).
The acylating agent may be added directly to a reaction mixture as a solid material (e.g., a powder) or dissolved in a suitable organic solvent such as acetone, N,N-dimethylformamide (DMF), ethanol, or methyl pyrrolidone. The total quantity of acylating agent depends on the extent of modification required or desired. As discussed above, the quantity of acylating agent required should generally satisfy the weight ratio of acylating agent to wet tissue of approximately 0.004:1 or less (i.e., less than or equal to 0.4% of wet tissue weight), preferably less than 0.003:1 (i.e., less than 0.3%), and most preferably from about 0.002:1 to about 0.001:1 (i.e., between 0.2% and 0.1%).
Using any of the above reagents, the acylation reaction (i.e., the amine modifying reaction) generally proceeds within a pH range of 7 to 11, although it is preferably carried out at a mildly basic pH (for example, pH 8-10, and, more preferably 8.5-9) to increase the reaction speed and reduce the processing time. For acylating the free amine groups on proteins associated with tissues, an acylation buffer at physiological pH is preferably utilized.
The reaction time for acylation of collagen will vary according to a number of factors including, for example, the amount of collagen to be acylated, the type of acylating agent, the pH, and the temperature of the reaction mixture. In addition, the method of addition of the acylating agent to the collagen composition will affect the reaction time. For example, addition of the acylating agent as a solid or in an appropriate solution will increase and decrease the reaction time, respectively. The reaction time is generally slower if the acylating agents are added as solids or powders.
In general, the acylation reaction should proceed to completion within a time ranging from about 30 seconds to about 10 minutes following each addition, preferably from about 1 to about 5 minutes. The reaction will generally occur at any reaction temperature between about 0xc2x0 C. to about 45xc2x0 C., but is preferably effected at about 20xc2x0 C. to about 37xc2x0 C., and especially at room temperature (about 25xc2x0 C.) for greatest convenience.
In addition to the above-mentioned methods, the acylation methods provided in any of DeVore et al. and Kelman et al., U.S. Pat. Nos. 5,492,135; 5,104,957; 5,201,764; 4,969,912, 5,332,802 and 5,480,427, may also be utilized, and these patents are incorporated herein by reference.
In one embodiment of the invention, following treatment with the acylation agent, the fine macerated, dispersed dermis is preferably subjected to additional static homogenization, subjected to filtration though a fine mesh screen, recovered by centrifugation, mixed to ensure homogeneity, and filled into syringes for clinical administration. Once the dispersed tissue matrix is prepared, it may be subjected to additional treatments to introduce intramolecular and/or intermolecular crosslinks to further stabilize the dispersion, rending it even more resistant to enzymatic digestion. Such treatments may include glutaraldehyde, EDAC {1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide}, and other standard chemicals and reagents commonly used to crosslink collagen.
The injectable, dispersed collagen matrix compositions produced by the methods of the invention have a variety of clinical uses, particularly as therapeutic or cosmetic implants. For example, the compositions of the invention may be used as intradermal implants to augment soft connective tissue or to correct skin defects such as wrinkles and scars. The compositions are also useful as injections into vocal folds to treat dysphonia or glottic insufficiency, as injections into submucosa of the urethra to treat urinary incontinence due to intrinsic sphincter deficiency, and as injectable drug delivery systems for localized drug administration. Other uses include replacement, augmentation, or other alteration of the condition of connective tissue, for example, in the form of a material for skin grafts, matrix substances or components for cell seeding and grafting, a material matrix for tissue xe2x80x9cputtyxe2x80x9d or filler, and the like.
The features and other details of the invention will now be more particularly described and pointed out in the following examples describing preferred techniques and experimental results. These examples are provided for the purpose of illustrating the invention and should not be construed as limiting.